Throughout this application various publications are referred to in parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications, and of all books, patents and patent application publications referred to herein, are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.
Bacterial infection and sepsis are the most common causes of death in the intensive care unit, annually claiming >225,000 victims in the U.S. alone. The pathogenesis of sepsis remains poorly understood, but is attributable to dysregulated systemic inflammation partly mediated by macrophages/monocytes (1, 2). Macrophages/monocytes are equipped with pattern recognition receptors [PRRs, such as the Toll-like receptors (TLRs), TLR2, TLR4, and TLR9] (3-5), and can bind various pathogen-associated molecular patterns (PAMPs, such as bacterial peptidoglycan, endotoxin, and CpG-DNA) (6-9). Consequently, these innate immune cells release a wide array of early proinflammatory cytokines such as TNF, IL-1 and IFN-γ 10-13. Excessive release of early cytokines contributes to the pathogenesis of LSI 12, 14-16. However, the therapeutic windows for these early mediators are relatively narrow (FIG. 1), prompting the search for other “late” proinflammatory mediators that may offer better therapeutic opportunities.
A decade ago, this laboratory made the seminal finding that high mobility group box-1 (HMGB1) was released from macrophages or monocytes in response to exogenous PAMPs (e.g., endotoxin or CpG-DNA) (17, 18) or endogenous cytokines (e.g., TNF or IFN-γ) (17, 19). Upon binding to the receptor for advanced glycation end products (RAGE), TLR2 or TLR4 (20-22), HMGB1 induces the expression of various cytokines, chemokines, and adhesion molecules (20, 21, 23-29). Consequently, extracellular HMGB1 functions as an alarmin signal to alert, recruit and activate innate immune cells (30-34), thereby sustaining rigorous and potentially injurious LSI. During endotoxemia or sepsis (induced by cecal ligation and puncture, CLP), circulating HMGB1 increased to plateau levels between 24-36 h (FIG. 1) (17, 35). This late appearance precedes the onset of animal lethality, and distinguishes HMGB1 from TNF and other early cytokines (36). The pathogenic role of HMGB1 was inferred from the observations that HMGB1-neutralizing antibodies (17, 35, 37) and inhibitors (e.g., tanshinones, ethyl pyruvate, nicotine, stearoyl lysophosphatidylcholine, epigallocatechin-3-gallate, nicotine, choline, GTS-21, and spermine) (17, 38, 39, 39-47) confer protection against lethal endotoxemia and sepsis, even when the first dose of antidote was given 24 h after CLP—a time point when mice had developed clear signs of sepsis, including lethargy, diarrhea, and piloerection. Conversely, administration of exogenous HMGB1 to mice recapitulated many clinical manifestations of sepsis, including fever 48, derangement of intestinal barrier function 49, and tissue injury (50-53). Collectively, these data establish HMGB1 as a critical “late” mediator of sepsis with a wider therapeutic window (36, 54-56) (FIG. 1).
On one hand, early cytokines TNF and IFN-γ can directly stimulate macrophages or monocytes to release HMGB1 (17, 19), thereby contributing to LPS-induced HMGB1 release. On the other hand, these early cytokines also alter the expression of liver-derived APPs, which may then participate in the regulation of HMGB1 release. For instance, TNF, IL-1, IL-6 57, 58 and IFN-γ (58) inhibited the hepatic expression of a negative APP, fetuin-A, which functions as a negative regulator of HMGB1 release during cerebral ischemia (59), endotoxemia and sepsis (58). However, the possible roles of other positive APPs in the regulation of HMGB1 release have not been identified.
In 1976, serum amyloid A (SAA) was first isolated from human serum as a 12 kDa protein (60) that shared identical N-terminal amino acid sequence with the previously characterized 8.5 kDa tissue amyloid A (AA) protein. Subsequently, it was found that exogenous endotoxin (61, 62) or endogenous cytokines (e.g., TNF, IL-1β and IFN-γ) (63-67) can all induce SAA expression in both hepatocytes and extrahepatic cells, such as macrophages/monocytes (68), endothelial cells, smooth muscle cells, adipocytes (69), intestinal epithelial cells (70), and neurons (71, 72). Consequently, circulating SAA levels are dramatically elevated (up to 1000-folds) within 16-24 h of endotoxemia as a result of the de novo expression of early cytokine inducers and the subsequent synthesis of SAAs (61, 73, 74).
Clinically, SAA levels have been regarded as a hallmark/risk factor of many diseases including atherosclerosis (75), cardiovascular diseases (76, 77), Crohn's disease, ulcerative colitis (78), as well as LSI diseases (such as endotoxemia and sepsis) (79-81). Upon secretion, extracellular SAA acts as a chemoattractant for inflammatory cells such as macrophages/monocytes (82-84), T cells (85) and mast cells (86). Furthermore, it activates innate immune cells (e.g., macrophages, monocytes, neutrophils and mast cells) to produce various cytokines and chemokines (e.g., TNF, IL-1β, IL-6, IL-10, GM-CSF, IL-8, MCP-1, MIP-1α, and MIP-3α) (87-94). Finally, SAA stimulates non-immune cells to release other proinflammatory factors such as the group II secretory phospholipase A2 (sPLA2, in smooth muscle cells) (95, 96) and prostaglandins (in endothelial cells) (97). However, it is not known whether SAA occupies an important role in the regulation of HMGB1 release, thereby functioning as an “intermediate” (as opposed to TNF being “early” and HMGB1 being “late”) mediators of LSI (FIG. 1).
Extensive studies have revealed distinct functional domains in SAA that are specifically responsible for: 1) SAA secretion (i.e., the signal sequence, D1); 2) HDL binding (D2); 3) cellular adhesion (D3); 4) as-yet-undefined function (D4); 5) protease cleavage (between residues 76-77); and 6) cell activation (D5) (FIG. 2). For instance, the signal sequence directs pro-SAA to the endoplasmic reticulum, and is cleaved prior to extracellular secretion of the mature SAA (98). The α-helical D2 domain serves as the driving force for binding to high density lipoprotein (HDL) (99,100). Within the D2 domain, the first 10-15 residues are particularly critical for the amyloidosis of SAA (101), because deletion or mutation in this region impaired amyloid fibril formation (102, 103). The D3 domain contains YIGSD laminin-related and RGN fibronectin-related motifs, and can inhibit T cell and platelet adhesion to extracellular matrixes (104, 105). At position 76-77 lies a cleavage site for the leukocyte-derived proteases, which break the serine-leucine bond (106-108) to liberate the 8.5-kDa amyloid A (AA) and 3.5-kDa (D5) fragments. The accumulation of the AA precipitates the formation of amorphous amyloid fibril deposits—amyloidosis in peripheral tissues with progressive loss of organ function. On the other hand, the 3.5-kDa fragment may be proinflammatory, because a synthetic peptide corresponding to residues 98-104 stimulates human CD4 T cells to produce IFN-γ (109).
The present invention addresses the need for improved therapies, based on SAA, to combat sepsis and endotoxemia.